Granulocyte chemotactic protein

ABSTRACT

This invention is directed to a new family of mammalian chemokine proteins, which have been designated granulocyte chemotactic protein-2 (GCP-2) proteins, and includes mammalian GCP-2, sequence-related variants of mammalian GCP-2, and distinct peptide fragments of GCP-2.

FIELD OF THE INVENTION

The present invention relates to chemotactic cytokine proteins, alsoknown as chemokines. In particular, the present invention relates to anovel family of granulocyte chemotactic proteins.

BACKGROUND OF THE INVENTION

An essential feature of the immune system is the mechanism of neutrophilactivation and phagocytosis, a process whereby foreign infectingparticles are enveloped by phagocytes which function to excrete suchparticles from a cell. Chemotactic cytokines have been found to play asignificant role in stimulating the migration of neutrophils to inflamedsites of infection, in order to kill invading microorganisms and allowphagocytosis to occur.

The cytokine family includes a number of structurally related proteins.Low molecular weight cytokines can be grouped into two subfamilies basedon differences in protein structure and in chromosomal location of genescoding for the cytokine (Oppenheim et al., 1991, Annu. Rev. Immunol.,9:617; Wolpe et al., 1989, FASEB J., 3:2565). One subfamily ofchemotactic proteins, the genes for which are located on humanchromosome 17, possess two adjacent cysteine residues (C-C) in theiramino-terminal protein sequence. Members of the other subfamily areencoded within human chromosome 4, and have a pair of cysteinesseparated by an amino acid (C-X-C). The first subfamily predominantlyincludes a number of monocyte chemotactic proteins (MCP) and the secondsubfamily is composed of granulocyte chemotactic proteins (GCP)including interleukin-8 (IL-8) and melanoma growth stimulating factor(GRO).

Some of the cytokines have been characterized by both in vitro and invivo studies (Oppenheim and Wolpe, supra). For example, IL-8 has beenreported to activate neutrophils leading to changes in cell shape,chemotaxis, degranulation, adherence to endothelium, increased vascularpermeability and trans-endothelial emigration into tissues (Van Damme,1991, Interleukin-8 and Related Molecules: The Cytokine Handbook, 201).GRO was first reported as a molecule involved in tumorigenesis (4). Onthe basis of its structural similarity with IL-8, GRO was later alsocharacterized as a neutrophil activating protein (NAP). However, thebiological functions of other identified proteins of this family, suchas inflammatory protein-10 (IP-10), are not well understood (Oppenheimand Wolpe, supra).

Due to the importance of chemokines in the inflammatory process, itwould be desirable to provide fully characterized proteins belonging tothe chemokine family to enable the development of chemokine agonisticand antagonistic therapeutics for the treatment of inflammatoryconditions.

Accordingly, it is an object of the present invention to provide novelchemotactic proteins.

SUMMARY OF THE INVENTION

A novel mammalian chemokine has been isolated and is designated hereingranulocyte chemotactic protein-2 or GCP-2, a chemokine which isselectively chemotactic for granulocytes and, further, stimulatesgranulocytes to secrete proteases such as gelatinase B. Sequence-relatedvariants of GCP-2 have also been identified. GCP-2 and suchsequence-related variants of GCP-2 are herein referred to as GCP-2proteins. Also encompassed by the term "GCP-2 proteins" are distinctpeptide fragments of GCP-2.

The present invention thus provides, in one of its aspects,substantially pure mammalian granulocyte chemotactic protein-2 (GCP-2)proteins, collectively comprising mammalian GCP-2, sequence-relatedvariants thereof and distinct peptide fragments.

In another aspect of the present invention, there is provided anisolated polynucleotide encoding a GCP-2 protein. There is also provideda recombinant vector having incorporated therein a polynucleotideencoding a GCP-2 protein, and a host cell that has been engineeredgenetically to produce a GCP-2 protein having incorporated expressiblytherein heterologous DNA encoding said GCP-2 protein. Further, arecombinant method for producing a GCP-2 protein comprising the step ofculturing cells which have incorporated expressibly therein apolynucleotide encoding said protein is provided.

In another aspect of the invention, there is provided a pharmaceuticalcomposition comprising a therapeutically effective amount of a GCP-2protein and a pharmaceutically acceptable carrier.

These and other aspects of the present invention will be described byreference to the following figures in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chemotactic activity of HPLC-isolated GCP-2proteins;

FIG. 2 identifies the amino acid sequence of human and bovine GCP-2(Seq. ID NOs: 14 and 15), and GCP-2 variants (Seq. ID NOs: 2-5 and16-19) and GCP-2 fragments (Seq. ID NOs: 6-13 and 20-24) of human andbovine GCP-2, thereby providing a comparison between the amino acidsequences of human and bovine GCP-2 proteins;

FIG. 3A illustrates an SDS-PAGE analysis of human GCP-2 and variantsthereof, and FIG. 3B identifies the amino-terminal sequence of each ofthese proteins (Seq. ID NOs: 2-5);

FIG. 4A illustrates an SDS-PAGE analysis of bovine GCP-2 and variantsthereof, and FIG. 4B identifies the amino-terminal sequence of each ofthese proteins (Seq. ID NOs: 16-19);

FIG. 5 graphically compares the granulocyte chemotactic activity ofbovine GCP-2 with other chemokines using a microchamber granulocytemigration assay;

FIG. 6 illustrates a zymographic SDS-PAGE analysis of the dose-dependentstimulation of gelatinase B release from granulocytes by GCP-2 ascompared to interleukin-8;

FIG. 7 graphically illustrates lack of GCP-2 reactivity in an IL-8radioimmunoassay, and granulocyte chemotactic activity isolated fromhuman tumour cells and fractionated by cation-exchange chromatography;and

FIG. 8 illustrates the amino acid sequences of various chemokines (Seq.ID NOs: 14, 15 and 24-45).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel family of mammalian chemokineproteins, herein designated GCP-2 proteins, which comprises mammalianGCP-2, sequence-related variants of mammalian GCP-2 and distinct peptidefragments of GCP-2.

Mammalian GCP-2, also referred to herein as GCP-2 per se, is defined asa naturally occurring parent protein within the GCP-2 family ofproteins. Mammalian GCP-2 may be derived from any mammalian source andthus includes not only human GCP-2, having the amino acid sequence (Seq.ID NO: 14) identified as HU-GCP-2 in FIG. 2, but also other naturallyoccurring mammalian forms such as rat, murine, feline, equine, caprine,porcine and bovine GCP-2. The amino acid sequence (Seq. ID NO: 15) ofbovine GCP-2, for example, is identified in FIG. 2 as BO-GCP-2.

The term "sequence-related variants" is meant to encompass thosevariants of GCP-2 which share substantial sequence homology with theirnative GCP-2 parent protein, i.e. no less than about 80% homologybetween parent and variant. GCP-2 variants may be naturally occurring orsynthetically-derived and may vary from the parent mammalian GCP-2 byamino acid deletion, insertion or substitution. Amino acid changes mayoccur within the amino acid sequence of the variant at either terminusthereof, i.e. amino-terminus or carboxy-terminus, or at an internallocation within the sequence of the variant. Variants in accordance withthe present invention will retain cellular recognition, i.e. GCP-2receptor binding ability, while GCP-2 biological activity includinggranulocyte chemotactic activity and protease secretion activity may ormay not be retained. As such, GCP-2 variants may have utility not onlyas agonists of GCP-2, but also as antagonists of GCP-2 which arerequired to be at least partially deficient in biological activity whilemaintaining sufficient homology to native GCP-2 to permit recognition invivo.

Also included within the meaning of the term "GCP-2 proteins" aredistinct peptide fragments of GCP-2. The term "distinct" is meant toencompass those fragments of GCP-2 which comprise an amino acid sequencethat is unique to GCP-2. As with GCP-2 variants, such fragments willretain cellular recognition, i.e. GCP-2 receptor binding ability, butmay or may not retain other biological activities of GCP-2 such asgranulocyte chemotactic activity and protease secretion activity.Accordingly, GCP-2 fragments may also be useful as agonists andantagonists of GCP-2.

Mammalian GCP-2 can be obtained by culturing a mammalian cell samplewhich is stimulated to produce cytokines. Using a series of purificationtechniques, substantially pure GCP-2 can then be isolated from the cellsample. As used herein, the term "substantially pure" refers to GCP-2purified to homogeneity as indicated by the electrophoretic technique ofSDS-PAGE, and by amino acid sequence analysis. More specifically, theterm "substantially pure" refers to GCP-2 which contains less than about5% by weight of contaminating protein, including, for example,interleukin-8, and preferably less than about 2% by weight of proteincontaminants. The purity of the present GCP-2 may be further defined interms of biological activity, i.e. the biological activity of thepresent substantially pure GCP-2 was not reduced in the presence of IL-8antibody.

In a specific embodiment of the present invention, GCP-2 was producedand isolated from tumour cells, i.e. human tumour cells. The tumourcells were grown in a minimum essential medium. Monolayers of the cellswere then induced to secrete cytokine proteins, for example, by additionof a cytokine such as interleukin-1β. Alternatively, a cytokine orcytokine mixture derived from mitogen-stimulated mononuclear cells canbe used. Following a suitable induction period, the cells, optionallywashed to remove the inducer, were incubated for up to about 96 hours.Granulocyte chemotactic activity was then isolated from the medium andpurified using a combination of conventional purification techniques.Initially, the chemotactic fraction of the medium was partially purifiedusing the technique of adsorption to controlled pore glass. Furtherpurification of the chemotactic activity was attained using antibodyaffinity chromatography or heparin-Sepharose chromatography.Cation-exchange fast protein liquid chromatography (FPLC) was thenutilized to purify further the chemotactic activity obtained from theantibody affinity or heparin-Sepharose chromatography step. A finalpurification step consisted of reverse phase high pressure liquidchromatography (HPLC) in which the chemotactic activity was purified tohomogeneity as determined by elution of single peaks, and by SDS-PAGE.

In another embodiment of the present invention, GCP-2 was obtained fromnon-tumour cells, i.e. bovine kidney cells. The method was substantiallythe same as the method used to obtain GCP-2 from tumour cells with theexception that the cells were stimulated by incubation with thestimulant, phorbol 12-myristate 13-acetate, for about 48 hours.Granulocyte chemotactic activity was then isolated using thepurification techniques described above.

GCP-2 may also be obtained using established techniques of recombinantDNA technology. Such techniques generally involve expression ofGCP-2-encoding DNA in a genetically engineered host cell. With knowledgeof the amino acid sequence of mammalian GCP-2, DNA encoding GCP-2 may besynthetically produced, for example, using an automated synthesizer. Thesynthesis is generally conducted by the successive 3' to 5' coupling ofappropriately protected nucleotide reagents followed by recovery of thedesired deprotected polynucleotide. Alternatively, the block ligationmethodology may be employed whereby oligonucleotide "blocks" are ligatedby overhang complementarity as described in Wosnick et al. in Gene,1989, 76:153. Sequences obtained by de novo synthesis may be amplifiedusing the polymerase chain reaction (PCR) as described in Barnett et al.in Nucl. Acids Res., 1990, 18(10):3094. Such synthetically-derived formsof GCP-2 DNA are herein referred to as "isolated" polynucleotides whichare free from association with DNA encoding other proteins.

Upon obtaining the desired GCP-2-encoding DNA, the DNA is inserted intoa suitable expression vector which is subsequently introduced into anappropriate host cell for expression. Such transformed host cells areherein characterized as having the GCP-2 DNA incorporated "expressibly"therein. Suitable expression vectors are those vectors which will driveexpression of the inserted GCP-2 DNA in the selected host. Typically,expression vectors are prepared by site-directed insertion therein ofthe GCP-2 DNA construct. The DNA construct is prepared by replacing acoding region, or a portion thereof, of a gene native to the selectedhost, or of a gene originating from a virus infectious to the host withthe GCP-2 DNA. In this way, regions required to control expression ofthe GCP-2 DNA which are recognized by the host, including a region 5' ofthe GCP-2 DNA to drive expression and a 3' region to terminateexpression, are inherent in the DNA construct. To allow selection ofhost cells stably transformed with the expression vector, a selectionmarker is generally included in the vector which takes the form of agene conferring some survival advantage on the transformants such asantibiotic resistance.

Expression of GCP-2 DNA, for example, in yeast can be achieved using theexpression-controlling regions of the yeast genes for proteins such asalcohol dehydrogenase, melibiase and many others. In Aspergillusspecies, GCP-2 production can be driven by the regions which control theexpression of alcohol dehydrogenase and aldehyde dehydrogenase inAspergillus nidulans, glucoamylase in A. niger, and amylase in A.oryzae. Further, the expression controlling regions of baculovirus genesmay be utilized in insect cell-based production of GCP-2. For mammaliancell-based production of GCP-2, expression controlling regionsassociated with SV40 and CMV viruses are suitable for use. The controlregions that regulate metallothionine in mammalian cells are alsosuitable.

A variety of hosts are suitable for use in expressing GCP-2 DNA,particularly in view of the fact that GCP-2 does not containN-glycosylation sites. Thus, both prokaryotic and eukaryotic host cellsmay be used. Suitable prokaryotic hosts include those hosts which arecapable of secreting the GCP-2 product, for example, bacterial hostssuch as E. coli and B. subtilis. Suitable eukaryotic hosts includefungal sources such as Aspergillus nidulans and Aspergillus niger andyeast sources such as Saccharomyces. Eukaryotic cell systems includingmammalian cell systems, such as Chinese hamster ovary cells (CHO cells)for example of K1 lineage (ATCC CCL 61) including the Pro5 variant (ATCCCRL 1281); fibroblast-like cells derived from SV40-transformed AfricanGreen monkey kidney of the CV-1 lineage (ATCC CCL 70), of the COS-1lineage (ATCC CRL 1650) and of the COS-7 lineage (ATCC CRL 1651); murineL-cells, murine 3T3 cells (ATCC CRL 1658), murine C127 cells, humanembryonic kidney cells of the 293 lineage (ATCC CRL 1573), humancarcinoma cells including HeLA lineage (ATCC CCL 2) and neuroblastomacells of the lines IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10) andSK-N-SH (ATCC HTB 11), are also suitable hosts.

As an alternative to isolating GCP-2 from biological samples or usingrecombinant techniques to produce GCP-2, GCP-2 may also be obtainedusing automated protein synthesis techniques. Such techniques are alsowell-established in the art and are currently applicable to proteins andpeptides comprising up to about 100 amino acid residues. Mammalian GCP-2is well within this limit as it comprises 75 amino acid residues. Onesuitable synthetic method is the solid-phase method. Generally, thesolid-phase method includes the step-wise addition of protected aminoacids to a growing peptide chain which is linked to an insoluble matrix,such as polystyrene beads. Initially, the carboxy-terminal amino acid ofthe peptide is linked to the matrix, the amino acid is deprotected usingtrifluoroacetic acid and the next amino acid in the chain is added tothe mixture in combination with a coupling agent such asdicyclohexylcarbodiimide (DCC). Once completed, the full-length peptideis released from the matrix by the addition of hydrofluoric acid, andany remaining protecting groups, i.e. protecting groups put on anypotentially reactive amino acid side chains, are removed to yield theGCP-2 product.

Sequence-related variants of GCP-2 may be isolated from biologicalsamples in the manner described for the isolation of GCP-2 itself. Inspecific embodiments of the present invention, naturally occurringvariants of GCP-2 were isolated from human tumour cell culture andbovine kidney cell culture, and subsequently purified by reverse-phaseHPLC. Amino-terminal sequence analysis in both cases revealed fulllength mammalian GCP-2 sequences as well as truncated sequence-relatedvariant sequences. These variants differed from the parent GCP-2molecule in amino acid sequence at the amino-terminus. Variants of humanGCP-2 are identified by their amino-terminal sequences in FIG. 2 (Seq.ID NOs. 2-5). Specifically, these variants include deletions of 2, 5 and8 amino acids from the amino-terminal end of human GCP-2. Similarvariants exist for other forms of mammalian GCP-2, namely bovine GCP-2.Specifically, 6, 7 and 8 amino acid N-terminally-truncated variants ofbovine GCP-2 were isolated, the N-terminal amino acid sequences of whichare also illustrated in FIG. 2 (Seq. ID NOs: 16-19).

Alternatively, variants of GCP-2 may be designed and then syntheticallyproduced. One method commonly used to design suitable variants, i.e.variants having the desired biological activity, involves a step by stepreplacement of amino acids in the native protein to determine, usingconventional assays as described hereinbelow, which amino acids areresponsible for the receptor binding activity and other activities ofthe protein. Such amino acid replacement may be conducted at the nucleicacid level using site-directed mutagenesis, a conventional procedurewhich is generally set out by Kunkel et al. in Proc. Natl. Acad. Sci.,1985, 82:488. Site-directed mutagenesis may be used to alterspecifically one or more amino acids in the GCP-2 sequence by mutatingspecific nucleotide bases in GCP-2 DNA. Often it is more efficient toeffect the change in a small oligonucleotide segment rather than in thefull-length DNA, and once effected the mutated segment is ligated tounmutated oligonucleotides to form the full-length GCP-2 variant. Ondetermining the amino acids which are essential for receptor bindingactivity, these amino acids are retained and amino acids non-essentialfor binding may be deleted or replaced. Such amino acid deletions orreplacements may render a variant in which chemotactic activity, forexample, is either retained or suppressed, leading to the development ofagonistic or antagonistic proteins.

It will also be appreciated that GCP-2 variants, whether altered asdescribed above or not, may include conservative amino acidsubstitutions, i.e. substitution of native amino acids with amino acidshaving similar charges, for example, substitution of a basic amino acidsuch as lysine with another basic amino acid such as arginine.Conservative amino acid substitutes are well-known in the art.

Generally, regions of the chemokine family of compounds, includingGCP-2, which are important for receptor binding are those regions whichare highly conserved among the chemokines. In particular, those regions,surrounding and inclusive of the 4 cysteine residues common to thechemokines, as illustrated by a comparison of the amino acid sequences(Seq. ID NOs: 14, 15 and 24-45) of the members of this family (see FIG.8), have been determined to play a role in receptor binding. In the caseof GCP-2, these cysteine residues reside at positions 12, 14, 38 and 52,and thus, variants that retain sequence homology with the regions ofGCP-2 surrounding these positions will potentially retain receptorbinding activity.

The method described above for designing GCP-2 variants will also beuseful in the preparation of GCP-2 fragments. Once the regions importantfor receptor binding, chemotactic activity, and other activities of theprotein have been determined, agonistic or antagonistic fragments ofthese regions can be made. GCP-2 fragments may also be generated usingenzyme digestion with endoproteinases including for example, Lys-C,Glu-C, Asp-N, and Arg-C proteases. This method involves incubating GCP-2with a particular protease which cleaves a specific amino acid bond,i.e. the Lys-C protease cleaves GCP-2 at the C-terminus of a lysineresidue, to render a desired fragment, while the Asp-N protease cleavesat the N-terminus of an aspartic acid residue. Chemical digestion mayalso be used to generate suitable fragments in accordance with thepresent invention including, for example, incubation of the protein informic acid. Specific fragments resulting from GCP-2 enzymatic andchemical digestion are illustrated in FIG. 2 (Seq. ID NOs: 6-13 and20-24).

GCP-2 fragments may include a sequence which mimics a highly conservedregion of mammalian GCP-2 such as those regions containing one or moreof the four highly conserved cysteine residues described above. Forexample, one region which is conserved within mammalian GCP-2, and alsohighly conserved in other members of the chemokine family as shown inFIG. 8, is the sequence preceding and inclusive of the cysteine residuesat positions 12 and 14 of GCP-2. Specifically, this conserved regioncomprises the following amino acid sequence (Seq. ID NO: 1), as denotedby single-letter amino acid code:

    -ELRCXC-

wherein X represents any amino acid residue selected from the naturallyoccurring amino acids.

On determining the amino acid sequence of preferred GCP-2 variants andfragments, in accordance with techniques such as those described aboveto determine the regions essential for the various activities of GCP-2,recombinant methods or synthetic methods essentially as described forfull-length GCP-2 may be used to obtain the selected variants andfragments. Moreover, receptor binding activity, chemotactic activity andenzyme release activity of these GCP-2 proteins may be determined usingconventional assays, such as those described hereinbelow.

Competition binding assays to determine receptor binding activity may beconducted as generally described by Clark-Lewis et al., J. of Biol.Chem., 1991, 266:23128. Briefly, GCP-2 is iodinated with Enzymobeadreagent by incubation in a solution containing Na₁₂₅ I, potassiumphosphate, and D(+)-glucose. Labelled (iodinated) GCP-2 and anunlabelled test compound (i.e. a potential GCP-2 competitive bindingprotein) are incubated on ice with medium containing neutrophils, Hepesbuffer and bovine serum albumin for about 90 minutes. Following theincubation period, the neutrophils are separated from unboundradioactivity by centrifugation. The resulting supernatant is thenaspirated, and the radioactivity, i.e. displaced GCP-2, of the remainingcell sediment is determined on a gamma radiation counter. A testcompound that is found to bind receptor competitively with GCP-2, andthus has the ability to displace receptor-bound GCP-2, is a potentialGCP-2 agonist or antagonist.

As described in more detail in the specific examples herein, following adetermination that a GCP-2 protein has receptor binding activity, thechemotactic activity of the protein is determined. Chemotaxis may bemeasured under agarose. Briefly, this entails incubating granulocytes inGCP-2-containing agarose. Following a suitable incubation period at 37°C., the chemotactic effect of the test sample is determined bymicroscopically measuring the migration distance of the cells in theagarose. Spontaneous migration of cells. i.e. migration not influencedby the test sample, is accounted for by comparison to a negative controlsample which does not contain a chemoattractant. To ensure that themigration of cells is due to the chemoattractant, a positive control isused which contains a known chemoattractant such asN-formylmethionyl-leucyl-phenylalanine (fMLP).

Another assay that may be used to determine chemotactic activityincludes measuring the GCP-2 protein-induced migration of granulocytesin a microchamber. The lower compartment of the microchamber is loadedwith a GCP-2 sample to be tested, while the upper compartment is filledwith granulocyte-containing medium. The lower and upper compartments areseparated by a 5 μm pore-size polycarbonate membrane. Following asuitable incubation period, the membrane is removed, fixed and stained,and the chemotactic effect of the test sample is determined by scoringthe number of cells that migrated through the membrane. Againspontaneous migration is accounted for by comparison to a controlsample.

The ability to stimulate granulocytes to release enzymes is anothercharacteristic of GCP-2 that may be tested for in potential GCP-2agonist or antagonist compounds, and again is determined usingconventional assays for this purpose. One such assay is described inmore detail in the specific examples. It involves incubating neutrophilsin a GCP-2-containing medium for an appropriate time period. Followingincubation, the presence of gelatinase B activity is measured byzymography. Alternatively, the presence of elastase activity may bemeasured by addition of a chromogenic elastase substrate.

The therapeutic application of GCP-2 proteins can be realized using theabove-mentioned assays. Specifically, both agonistic and antagonisticGCP-2 proteins will be found to competitively bind a GCP-2 receptor.Further, agonistic proteins will exhibit chemotactic or secretoryactivity which is proportional to their recept or binding activity,while antagonistic proteins will be lacking in at least one ofchemotactic or enzyme secretory activity.

GCP-2 proteins, inclusive of GCP-2 per se and variants and fragmentsthereof, are also important as a tool for screening potential drugcandidates having agonistic or antagonistic activities. Such drugcandidates may or may not be GCP-2 variants or fragments as defiedherein, but rather may be any biologically- or chemically-derivedcompound that may possess the ability to bind a GCP-2 receptor.

In another aspect of the present invention, a pharmaceutical compositioncomprising a GCP-2 protein is provided. Such compositions may be in anysuitable administrable form including tablets, pills, capsules, powders,aerosols, suppositories, creams, lotions, ointments, skin patches,parenterals, oral liquids such as suspensions, solutions and emulsions,ophthalmic liquids and injectable liquids.

The present compositions are prepared by admixture of a GCP-2 proteinand a pharmaceutically acceptable carrier. As used herein, theexpression "pharmaceutically acceptable" means acceptable for use in thepharmaceutical and veterinary arts, and not being toxic or otherwiseunacceptable. The selection of carrier depends on the intended mode ofadministration of the composition. Thus, compositions to be administeredorally are prepared using substances that are suitably combined with theGCP-2 protein for oral ingestion. Such substances include, withoutlimitation, sugars, such as lactose, glucose and sucrose, starches suchas corn starch and potato starch; cellulose and derivatives thereof,including sodium carboxymethylcellulose, ethylcellulose and celluloseacetates; powdered tragacanth; malt; gelatin; talc; stearic acids;magnesium stearate; calcium sulfate; vegetable oils, such as peanutoils, cotton seed oil, sesame oil, olive oil and corn oil; polyols suchas propylene glycol, glycerine, sorbitol, mannitol and polyethyleneglycol; agar; alginic acids; water; isotonic saline and phosphate buffersolutions. Wetting agents, lubricants such as sodium lauryl sulfate,stabilizers, tabletting agents, anti-oxidants, preservatives, colouringagents and flavouring agents may also be present. Compositions to beadministered by injection are prepared using liquid carriers such asbuffered saline and physiological saline. Likewise, compositions forophthalmic administration are prepared in suitable liquid carriers suchas buffered or physiological saline. Creams, lotions and ointments maybe prepared for topical application using an appropriate base such as atriglyceride base. Such creams, lotions and ointments may also contain asurface active agent.

In treating an inflammatory condition in a mammal, a therapeuticallyeffective amount of the present composition is administered thereto. Theterm "mammal", as used with respect to such treatment and as usedelsewhere herein, is meant to encompass, without limitation, humans,domestic animals such as dogs, cats, horses, cattle, swine, sheep, goatsand the like, as well as wild animals. Further, as used herein, the term"therapeutically effective amount" is an amount of the compositionindicated for treatment of the condition, for example an inflammatorycondition, while not exceeding an amount which may cause significantadverse effects.

Embodiments of the present invention are described in the followingspecific examples which are not to be construed as limiting.

EXAMPLE 1 Production and Purification of Human and Bovine GCP-2

Human MG-63 osteosarcoma cells (ATCC CRL 1427) and bovine MDBK (MadinDarby bovine kidney) cells (ATCC CRL 6071) were separately grown inEagle's minimum essential medium (EMEM, Gibco, Paisley, Scotland) withEarle's salts containing 10% fetal calf serum (Gibco).

Confluent monolayers (175 cm², Nunc, Roskilde, Denmark) of MG-63 cellswere stimulated in serum-free medium with silicic acid-purifiedleukocyte-derived human cytokine preparation from mononuclear cellsstimulated with lipopolysaccharide (E. coli 0111.B4, Difco, Detroit,Mich.) and concanavalin A (Calbiochem, San Diego, Calif.) (Van Damme etal., 1988, J. Exp. Med. 167:1364). After 5 hours, the induction mediumwas removed and the cells were incubated with serum free medium for48-96 hours.

MDBK cell cultures were washed and incubated with 25 ml serum-freemedium containing 10 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma,St. Louis, Mo.) for 48 h.

Conditioned medium of three consecutive inductions of MG-63 cells andMDKB cells were respectively pooled, concentrated and partially purifiedby adsorption to controlled pore glass (CPG-10-350, Serva, Heidelberg,Germany). Chemotactic activity was eluted with 0.3M glycine/HCl, pH 2.0.The CPG-eluate was neutralized and further purified by antibody affinitychromatography using a polyclonal antibody to CPG-purified,fibroblast-derived cytokines (Van Damme et al., 1987, Eur. J. Biochem.,168:543). Alternatively, the eluate was loaded onto a heparin-Sepharose(CL-6B, Pharmacia, Uppsala, Sweden) column in 50 mM Tris, 50 mM NaCl, pH7.4. After washing with this equilibration buffer, the chemotacticactivity was eluted in a linear NaCl gradient (0.05-2M) in 50 mM Tris,pH 7.4. The heparin-Sepharose- or antibody-derived chemotactic activitywas further purified by Mono S cation-exchange fast protein liquidchromatography (FPLC, Pharmacia) in 50 mM formate, pH 4.0. Proteins wereeluted in a linear NaCl gradient (0-1M) in 50 mM formate, pH 4.0. Thefinal purification step consisted in C8 RP-HPLC of the chemotactic FPLCfractions using an HPLC system comprising a model 2150 HPLC pump, amodel 2152 system controller, and a model 2151 variable wavelengthmonitor (LKB, Broma, Sweden). One ml FPLC fractions were injected onto a220×2.1 mm C8 Aquapore RP-300 column (Applied Biosystems Inc., FosterCity, Calif.) equilibrated with 0.1% trifluoroacetic acid (TFA) in H₂ O(Solvent A) and eluted with an acetonitrile gradient (Solvent B: 80% CH₃CN; 0.1% TFA in H₂ O) at 0.4 ml/min.

FPLC- and HPLC- derived fractions were checked for purity by SDS-PAGE ona linear gradient (10-25%) polyacrylamide gel by silver staining. Therelative molecular mass markers (Bio-Rad Laboratories, Richmond, Calif.)used were phosphorylase b (M_(r) 92,500), BSA (M_(r) 66,200), ovalbumin(M_(r) 45,000), carbonic anhydrase (M_(r) 31,000), soybean trypsininhibitor (M_(r) 21,500), lysozyme (M_(r) 14,400) and the low relativemolecular mass marker, aprotinin (M_(r) 6,500) (Pierce Chemical Co.,Rockford, Ill.).

FIG. 1 illustrates that upon purification to homogeneity by HPLC, thehuman GCP-2 activity dissociated into multiple peaks. Four 6 kDaproteins eluting in the corresponding active fractions were visualizedby SDS-PAGE (FIG. 3A) and were identified as GCP-2 and GCP-2 variants byNH₂ -terminal sequence analysis (about 30 cycles for each form). Exceptfor NH₂ -terminal truncation no differences were observed between thesequences of these proteins (Seq. ID NOs: 2-5). This results in GCP-2forms (variants) that are missing two (fraction 60), five (fraction 56)and eight (fraction 54) amino acids, respectively (FIGS. 2 and 3B).

EXAMPLE 2 Generation and Sequencing of Human GCP-2 Proteins

In order to identify the chemotactic proteins, the NH₂ -terminal acidsequence was determined by Edman degradation on a pulsed liquid(477A/120A) amino acid sequencer (Applied Biosystems) with on-linePTH-amino acid analysis. Cysteine residues were determined by on filterreduction and modification with tributylphosphin and 4-vinylpyridine(Aldrich Chemical Company, Inc., Wis.) (Andrews et al., 1987, Anal.Biochem., 161:524).

To extend the sequence information, internal peptide fragments (Seq. IDNOs: 6-12) were prepared using different proteolytic enzymes.Chemotactic protein (4 μg) was incubated with 0.2 μg of enzyme in thesuitable incubation buffer and peptide fragments were separated on a C8Aquapore RP-300 column as described for the final purification of thechemotactic factors. Endoproteinases (sequencing grade; BoehringerMannheim; Mannheim; Germany) used were: Lys-C (37° C., 18 h in 25 mMTris-HCl buffer, pH 8.5, 1 mM EDTA), Arg-C (37° C., 18 h in 90 mMTris-HCl buffer, pH 7.6, 8.5 mM CaCl₂, 5 mM DTT, 0.5 mM EDTA), Asp-N(37° C., 18 h in 50 mM sodium phosphate buffer, pH 8.0) and Glu-C (25°C,. 18 h in 25 mM ammonium carbonate buffer pH 7.8). All peptides weresequenced as described above.

Alternatively, chemical digestion of the proteins was performed in 75%formic acid at 37° C. for 50 h. After the formic acid digestion, peptidefragments (Seq. ID NO: 13) were dried on the cartridge filter of theprotein sequencer. A solution of o-phthalaldehyde (Fluoropa, Pierce) and2-mercaptoethanol in acetonitrile was added and the fragments wereincubated for 10 min in a continuous triethylamine flow in order toblock all peptide chains except for the one starting with an NH₂-terminal proline (Brauer et al., 1984, Anal. Biochem. 137:134).Remaining reagents were washed away with ethyl acetate andn-butylchloride.

After endoproteinase Lys-C digestion, four internal fragments could besequenced (FIG. 2, Seq. ID NOs: 8-11). Endoproteinase Asp-Nfragmentation yielded a fragment (Seq. ID NO: 12) which confirmedsequence information obtained by NH₂ -terminal sequencing and byendoproteinase Lys-C digestion. Cleavage of the protein withendoproteinase Glu-C yielded an N-terminal fragment (Seq. ID NO: 6) anda fragment (Seq. ID NO: 7) overlapping the gap between the Lys-Csequence stretches. The carboxy-terminal residues were determined byformic acid cleavage of the Asp-Pro bond to yield a fragment of Seq. IDNO: 13). In this case, all peptides obtained, except those starting witha proline, were blocked at the NH₂ -terminus with o-phthalaldehydeallowing determination of the COOH-terminal sequence of human GCF-2without purification of the formic acid digest.

Alignment of the sequence from the multiple GCP-2 fragments resulted inthe complete elucidation of the primary structure of this novelgranulocyte chemotactic protein (Seq. ID NO: 14). Most residues havebeen confirmed several times by repeated sequencing of overlappingfragments. The sequence of human GCP-2 allows classification of themolecule as a member of the chemokine family, based on the conservationof four cysteine residues. The protein is most related to human ENA-78(Seq. ID NO: 31) (Walz et al., 1991, J. Exp. Med. 174:1355) (77%similarity), whereas the relationship with human IL-8 (Seq. ID NO: 24)(31% similarity) is rather weak.

EXAMPLE 3 Isolation and Sequencing of Bovine GCP-2 Proteins

For the isolation of bovine GCP-2, serum-free conditioned medium ofbovine MDBK cells was processed as described for the isolation of humanGCP-2. FIG. 4A illustrates that, as in the case of human GCP-2, thebovine granulocyte chemotactic activity from FPLC dissociates after HPLCinto several 5 kDa protein bands. In particular, four protein peakseluted in the gradient at 31.5% (fraction 62), 32% (fraction 64), 32.5%(fraction 66) and 34% (fraction 72) acetonitrile, respectively.

NH₂ -terminal amino acid sequence analysis revealed that these proteinsonly differed in truncation at the NH₂ -terminus (FIG. 4B, Seq. ID NOs:16-19). The complete sequence of this bovine GCP-2 was deduced fromoverlapping COOH-terminal fragments obtained by endoproteinase Arg-Cdigestion (FIG. 2, Seq. ID NOs: 20-23) and confirmed by formic aciddigestion (Seq. ID NO: 24). The four cysteines typical for chemokinesare also conserved in bovine GCP-2 (Seq. ID NO: 15) which shares 67%homology with human GCP-2 (Seq. ID NO: 14) at the protein level.

EXAMPLE 4 Determination of GCP-2 Chemotaxis Activity

Heparinized human peripheral blood from single donors was suspended inhydroxyethyl starch (Plasmasteril, Fresenius AG, Bad Homburg, Germany)for 30 minutes to remove erythrocytes by sedimentation. Mononuclearcells and granulocytes were separated by gradient centrifugation (30minutes, 400×g) on Ficoll-sodium metrizoate (Lymphoprep, Nyegaard, Oslo,Norway). The total mononuclear cell fraction was used as a cell sourcefor monocyte chemotaxis experiments. Erythrocytes, present in thegranulocyte pellet, were eliminated by lysis in bidistilled water (30s). Purified granulocytes (98%) were obtained by centrifugation (30minutes, 20,000×g) in a Percoll (Pharmacia) gradient (d=1.054 g/ml).

Chemotaxis under agarose was measured according to the method of Nelsonet al., 1975, J. Immunol., 115:1650 and as previously described by VanDamme et al. 1988, J. Exp. Med., 167:1364. Mononuclear cells (10⁶ /well)or granulocytes (3×10⁵ /well) were exposed to serial dilutions of testsamples (10 μl/well) and to control medium (Hanks' Balanced Salt Sodium(HBSS) plus albumin). Purified human granulocyte (IL-8) and monocytechemotactic protein (MCP-1) at 10 U/ml (6,7), andN-formylmethionyl-leucyl-phenylalanine (fMLP, Sigma) at 10⁻⁷ M were usedas positive controls. After a 2-4 hour incubation at 37° C., cells werefixed and the migration distance was scored microscopically. Effectivemigration represents the difference between the migration distancetowards the test sample and the spontaneous migration distance towardsthe control medium. The titration end-point, corresponding to 1 U/ml,was calculated from a dilution resulting in the half-maximal effectivemigration distance as compared to that obtained with fMLP.

A second chemotaxis assay measured the migration of monocytes andgranulocytes using a microchamber (Neuro Probe Inc., Cabin John, Md.)technique (Falk et al., 1980. J. Immunol. Methods, 33:239). Human cellswere adjusted to a concentration of 1×10⁶ (granulocytes) or 2×10⁶(monocytes) cells/ml in HBSS supplemented with 1 mg/ml human serumalbumin to form a cell suspension. The lower compartment of themicrochamber was filled with dilutions of test samples (27 μl), whereasthe upper compartment contained 50 μl cell suspension. The twocompartments were separated by a 5 μm pore-size polycarbonate(polyvinyl-pyrrolidone-free for granulocytes) filter (Nucleopore,Pleasanton, Calif.). After incubation at 37° C. for 45 (granulocytes) or120 (monocytes) minutes, the filters were removed from the chambers,fixed and stained with Diff-Quick (Harleco, Gibbstown, N.J.). Finally,migrated cells of ten microscopic fields were counted. Optimalconcentrations of purified human MCP-1 (Van Damme et al., 1989, Eur. J.Immunol., 19:2367), IL-8 (Van Damme et al., 1988, supra) or fMLP (SigmaChemical Co., St. Louis, Mo.) were used as reference chemoattractants.The chemotactic index was calculated from the number of cells migratedto the test sample divided by the number of cells migrated to thecontrol medium.

HPLC-purified and NH₂ -terminally sequenced human GCP-2 (form I) andGCP-2 variants forms I-IV) were tested in the granulocyte microchamberassay to determine heir potency as granulocyte chemotactic factors. Theresults were as follows:

                  TABLE I                                                         ______________________________________                                        Comparison of human GCP-2 forms in stimulating neutrophil                     chemotaxis                                                                                                      Chemotactic                                 Chemokine                                                                              HPLC    NH.sub.2 -terminal                                                                        Conc.                                                                              index                                       form     fraction                                                                              sequence    (nN) mean ± SEM 9n).sup.a)                    ______________________________________                                        GCP-2-I  67      Seq. ID NO: 2                                                                             30   18.8 ± 4.9 (4)                           (75 AA)                      10    5.4 ± 1.3 (8)                                                        3     3.0 ± 1.1 (6)                                                        1     1.1 ± 0.0 (2)                           GCP-2-II 60      Seq. ID NO: 3                                                                             30   49.5 (1)                                    (73 AA)                      10   10.2 ± 2.5 (7)                                                        3     2.5 ± 0.4 (6)                                                        1     1.8 ± 0.3 (5)                           GCP-2-III                                                                              57      Seq. ID N O: 4                                                                            30   18.0 (1)                                    (70 AA)                      10    4.7 ±0 1.3 (6)                                                       3     2.4 ± 0.4 (6)                                                        1     2.2 ± 0.4 (5)                           GCP-2-IV 54      Seq. ID NO: 5                                                                             100  49.6                                        (67 AA)                      30   11.8 ± 1.8 (7)                                                        10    3.4 ± 0.5 (6)                                                        3     2.0 ± 0.4 (5)                           IL-8             KEL . . . (70 AA)                                                                         1    28.4 ± 7.8 (6)                           (72 AA +         SAK . . . (72 AA)                                                                         0.1   7.2 ± 1.2 (10)                          70 AA)                       0.01  3.0 ± 0.6 (4)                           ______________________________________                                         .sup.1) chemokine response expressed as average chemotactic index ±        standard error of the mean: n represents the number of determinations    

The results illustrated in Table I confirm that GCP-2 and the isolatednaturally occurring variants of GCP-2 are biologically active in themicrochamber assay. These GCP-2 forms stimulate neutrophil migration ina dose-dependent fashion.

Further, the biological activity of the four different forms of bovineGCP-2 (i.e. GCP-2 and N-terminal variants thereof) was also determinedusing the microchamber migration assay. FIG. 5 shows that bovine GCP-2and its variants have a comparable potency and efficacy on neutrophils.Although human cells were used as test substrate, the bovine moleculewas found to be as efficient as human IL-8 and human GCP-2 in thatmaximal chemotactic responses were similar.

The minimum effective dose of bovine GCP-2 was as low as 1 nM, and thus,it was at least as potent as human GCP-2 in this test using human cells.For human GCP-2 about 3 nM was necessary to observe a chemotacticresponse. When tested (concentration range of 1 to 100 nM) on humanmonocytes, human and bovine GCP-2, were found to be inactive.

EXAMPLE 5 Determination of GCP-2 Granulocyte Activation using an EnzymeRelease Assay

Release of gelatinase B was used as a parameter to measure granulocyteactivation. Purified granulocytes (1-3×10⁶ cells/ml) were stimulated inserum-free medium with test reagents for 15-45 minutes. Supernatantswere centrifuged to remove cells and gelatinase activity was determinedby SDS/PAGE zymography using gelatine as a substrate (Masure et al.,1990, Biochim. Biophys. Acta., 1054:317). Human IL-8 (Opdenakker et al.,1991, Lymphokine and Cytokine Research, 10:317) and fMLP were used aspositive controls for gelatinase B production.

Gelatinase activity was detected as unstained bands on a Coomassiebrilliant blue R-250 background. Quantitative determination ofgelatinase activity was achieved by computerized scanning densitometry.Gelatinase activity was expressed in "scanning units", representing thescanning area under the curves, which is an integration ratio that takesinto account both the brightness and the width of the substrate lysiszone.

Human and bovine GCP-2 were compared with IL-8 as neutrophil activating,proteins in an enzyme release assay. For this purpose secretion ofgelatinase B activity was measured by zymography. FIG. 6 illustratesthat the three molecules can dose-dependently stimulate gelatinase Brelease from human neutrophils within 15 minutes. The lower specificactivity of human GCP-2 when compared to bovine GCP-2 was also confirmedin this assay. The minimum effective concentration for GCP-2 is about 10nM suggesting that for gelatinase B release more chemokine is neededthan for neutrophil migration.

EXAMPLE 6 Crossreactivity of GCP-2 with IL-8

Pure natural IL-8 (Van Damme et al., 1988, supra) derived from humanperipheral blood leukocytes was used to prepare an IL-8 antibody ingoat. ¹²⁵ I-labelled IL-8 (2000 Ci/mmol) was purchased from Amersham(Buckinghamshire, United Kingdom). The radioimmunoassay for IL-8 wasperformed as described by Rampart et al., 1992, Lab. Invest., 66:512.Briefly, column fractions, IL-8 (natural) standard, ¹²⁵ I-IL-8 (1/1000)and IL-8 antibody (1/3000) were diluted in Tris-buffered saline, pH 7.4containing 1% bovine serum albumin and 0.2% gelatin. Column fractions orIL-8 standard (100 μl) were mixed with antibody (50 μl) and ¹²⁵ I-IL-8(50 μl) and were incubated for 18 hours at room temperature.Antibody-bound reactivity was precipitated by addition of protein-Abacterial absorbent. The detection limit of the assay was 0.1 ng/ml.

IL-8 has been detected in body fluids using the above-describedradioimmunoassay with labelled natural chemokine (Rampart et al., 1992,supra). In order to exclude crossreactivity of GCP-2 with IL-8, a batchof an MG-63 cell-derived chemokine mixture was fractionated bycation-exchange FPLC. FIG. 7 shows that in the microchamber assaygranulocyte chemotactic activity is detectable corresponding to GRO,GCP-2 and IL-8, eluting at 0.6, 0.7 and 0.85M NaCl, respectively (Proostet al., 1993, J. Immunol., 150:1000). However, with the radioimmunoassayfor IL-8 no immunoreactivity could be measured in the fractioncontaining GRO despite the fact that the assay was quite sensitive forIL-8 (detection limit of 0.1 ng/ml). GCP-2-containing FPLC fractions (5μg/ml) were found to contain a small amount of IL-8 immunoreactivity(about 20 ng/ml of IL-8 immunoreactivity); however, this may have beendue to contamination with authentic IL-8. From these results, it wascalculated that the IL-8 radioimmunoassay is at least 100-fold lessspecific for GCP-2 than for IL-8.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 45                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          MOLECULE TYPE: peptide                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4..6                                                            (D) OTHER INFORMATION: /note= "Xaa represents any amino                       acid"                                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GluLeuArgCysXaaCys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GlyProValSerAlaValLeuThrGluLeuArgCysThrCysLeuArg                              151015                                                                        ValThrLeuArgValAsnProLysThrIleGlyLysLeuGlnValPhe                              202530                                                                        ProAlaGly                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      ValSerAlaValLeuThrGluLeuArgCysThrCysLeuArgValThr                              151015                                                                        LeuArgValAsnProLysThrIleGlyLysLeuGlnValPheProAla                              202530                                                                        Gly                                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      ValLeuThrGluLeuArgCysThrCysLeuArgValThrLeuArgVal                              151015                                                                        AsnProLysThrIleGlyLysLeuGlnValPheProAla                                       2025                                                                          (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      GluLeuArgCysThrCysLeuArgValThrLeuArgValAsnProLys                              151015                                                                        ThrIleGlyLysLeuGlnValPheProAlaGly                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10..12                                                          (D) OTHER INFORMATION: /note= "Xaa was ambiguous or                           undetectable at time of determination; later                                  identified as "Cys""                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      ValSerAlaValLeuThrGluLeuArgXaaThrXaaLeuArgValThr                              151015                                                                        LeuArgValAsn                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      ValValAlaSerLeuLysAsnGlyLysGlnValCysLeuAspProGlu                              151015                                                                        (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      LeuGlnValPheProAlaGlyProGlnCysSerLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      GlnValCysLeuAspProGluAlaProPheLeuLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     LeuGlnValPheProAlaGlyProGlnCysSerLysValGluValVal                              151015                                                                        Ala                                                                           (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     ValGluValValAlaSerLeuLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4..6                                                            (D) OTHER INFORMATION: /label=xtx                                             /note= "x was ambiguous at time of determination"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 29..31                                                          (D) OTHER INFORMATION: /label=qxs                                             /note= "x was ambiguous at time of determination;                             later identified as "c""                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     GluLeuArgXaaThrXaaLeuArgValThrLeuArgValAsnProLys                              151015                                                                        ThrIleGlyLysLeuGlnValPheProAlaGlyProGlnXaaSerLys                              202530                                                                        ValGluValVal                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: C-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     ProGluAlaProPheLeuLysLysValIleGlnLysIleLeuAspSer                              151015                                                                        GlyAsnLys                                                                     (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     GlyProValSerAlaValLeuThrGluLeuArgCysThrCysLeuArg                              151015                                                                        ValThrLeuArgValAsnProLysThrIleGlyLysLeuGlnValPhe                              202530                                                                        ProAlaGlyProGlnCysSerLysValGluValValAlaSerLeuLys                              354045                                                                        AsnGlyLysGlnValCysLeuAspProGluAlaProPheLeuLysLys                              505560                                                                        ValIleGlnLysIleLeuAspSerGlyAsnLys                                             657075                                                                        (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     GlyProValAlaAlaValValArgGluLeuArgCysValCysLeuThr                              151015                                                                        ThrThrProGlyIleHisProLysThrValSerAspLeuGlnValIle                              202530                                                                        AlaAlaGlyProGlnCysSerLysValGluValIleAlaThrLeuLys                              354045                                                                        AsnGlyArgGluValCysLeuAspProGluAlaProLeuIleLysLys                              505560                                                                        IleValGlnLysIleLeuAspSerGlyLysAsn                                             657075                                                                        (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4..6                                                            (D) OTHER INFORMATION: /note= "Xaa was ambiguous at time                      of determination; later identified as "C..."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     GluLeuArgXaaValXaaLeuThrThrThrProGlyIleHisProLys                              151015                                                                        ThrValSer                                                                     (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 44..46                                                          (D) OTHER INFORMATION: /note= "Xaa was ambiguous at time                      of determination; later identified as "G..."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     ArgGluLeuArgCysValCysLeuThrThrThrProGlyIleHisPro                              151015                                                                        LysThrValSerAspLeuGlnValIleAlaAlaGlyProGlnCysSer                              202530                                                                        LysValGluValIleAlaThrLeuLysAsnGlyArgXaaVal                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 12..14                                                          (D) OTHER INFORMATION: /note= "Xaa was ambiguous at time                      of determination; later identified as "C..."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     ValArgGluLeuArgXaaValXaaLeuThrThrThrProGlyIleHis                              151015                                                                        ProLysThrValSerAspLeuGlnValIleAlaAlaGlyProGln                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 12..14                                                          (D) OTHER INFORMATION: /note= "Xaa was ambiguous at time                      of determination; later identified as "C..."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     GlyProValAlaAlaValValArgGluLeuArgXaaValXaaLeuThr                              151015                                                                        ThrThrProGlyIleHisProLysThrValSerAspLeuGlnValIle                              202530                                                                        AlaAlaGlyProGln                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     GluValCysLeuAspProGluAlaProLeuIleLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: C-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     IleValGlnLysIleLeuAspSerGlyLysAsn                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2..4                                                            (D) OTHER INFORMATION: /note= "Xaa was ambiguous at time                      of determination; later identified as "C..."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     GluValXaaLeuAspProGluAlaProLeuIleLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: C-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     LysIleValGlnLysIleLeuAspSerGlyLysAsn                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: C-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     ProGluAlaProLeuIleLysLysIleValGlnLysIleLeuAspSer                              151015                                                                        GlyLysAsn                                                                     (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     GluGlyAlaValLeuProArgSerAlaLysGluLeuArgCysGlnCys                              151015                                                                        IleLysThrTyrSerLysProPheHisProLysPheIleLysGluLeu                              202530                                                                        ArgValIleGluSerGlyProHisCysAlaAsnThrGluIleIleVal                              354045                                                                        LysLeuSerAspGlyArgGluLeuCysLeuAspProLysGluAsnTrp                              505560                                                                        ValGlnArgValValGluLysPheLeuLysArgAlaGluAsnSer                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     AlaSerValAlaThrGluLeuArgCysGlnCysLeuGlnThrLeuGln                              151015                                                                        GlyIleHisProLysAsnIleGlnSerValAsnValLysSerProGly                              202530                                                                        ProHisCysAlaGlnThrGluValIleAlaThrLeuLysAsnGlyArg                              354045                                                                        LysAlaCysLeuAsnProAlaSerProIleValLysLysIleIleGlu                              505560                                                                        LysMetLeuAsnSerAspLysSerAsn                                                   6570                                                                          (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     AlaProLeuAlaThrGluLeuArgCysGlnCysLeuGlnThrLeuGln                              151015                                                                        GlyIleHisLeuLysAsnIleGlnSerValLysValLysSerProGly                              202530                                                                        ProHisCysAlaGlnThrGluValIleAlaThrLeuLysAsnGlyGln                              354045                                                                        LysAlaCysLeuAsnProAlaSerProMetValLysLysIleIleGlu                              505560                                                                        LysMetLeuLysAsnGlyLysSerAsn                                                   6570                                                                          (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     AlaSerValValThrGluLeuArgCysGlnCysLeuGlnThrLeuGln                              151015                                                                        GlyIleHisLeuLysAsnIleGlnSerValAsnValArgSerProGly                              202530                                                                        ProHisCysAlaGlnThrGluValIleAlaThrLeuLysAsnGlyLys                              354045                                                                        LysAlaCysLeuAsnProAlaSerProMetValGlnLysIleIleGlu                              505560                                                                        LysIleLeuAsnLysGlySerThrAsn                                                   6570                                                                          (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     ValProLeuSerArgThrValArgCysThrCysIleSerIleSerAsn                              151015                                                                        GlnProValAsnProArgSerLeuGluLysLeuGluIleIleProAla                              202530                                                                        SerGlnPheCysProArgValGluIleIleAlaThrMetLysLysLys                              354045                                                                        GlyGluLysArgCysLeuAsnProGluSerLysAlaIleLysAsnLeu                              505560                                                                        LeuLysAlaValSerLysGluMetSerLysArgSerPro                                       657075                                                                        (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     GluAlaGluGluAspGlyAspLeuGlnCysLeuCysValLysThrThr                              151015                                                                        SerGlnValArgProArgHisIleThrSerLeuGluValIleLysAla                              202530                                                                        GlyProHisCysProThrAlaGlnLeuIleAlaThrLeuLysAsnGly                              354045                                                                        ArgLysIleCysLeuAspLeuGlnAlaProLeuTyrLysLysIleIle                              505560                                                                        LysLysLeuLeuGluSer                                                            6570                                                                          (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     AlaGluLeuArgCysMetCysIleLysThrThrSerGlyIleHisPro                              151015                                                                        LysAsnIleGlnSerLeuGluValIleGlyLysGlyThrHisCysAsn                              202530                                                                        GlnValGluValIleAlaThrLeuLysAspGlyArgLysIleCysLeu                              354045                                                                        AspProAspAlaProArgIleLysLysIleValGlnLysLysLeuAla                              505560                                                                        GlyAspGluSerAlaAsp                                                            6570                                                                          (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     AlaGlyProAlaAlaAlaValLeuArgGluLeuArgCysValCysLeu                              151015                                                                        GlnThrThrGlnGlyValHisProLysMetIleSerAsnLeuGlnVal                              202530                                                                        PheAlaIleGlyProGlnCysSerLysValGluValValAlaSerLeu                              354045                                                                        LysAsnGlyLysGluIleCysLeuAspProGluAlaProPheLeuLys                              505560                                                                        LysValIleGlnLysIleLeuAspGlyGlyAsnLysGluAsn                                    657075                                                                        (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 88 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     GluSerSerPheProAlaThrPheValProLeuProAlaAspSerGlu                              151015                                                                        GlyGlyGluAspGluAspLeuGlnCysValCysLeuLysThrThrSer                              202530                                                                        GlyIleAsnProArgHisIleSerSerLeuGluValIleGlyAlaGly                              354045                                                                        ThrHisCysProSerProGlnLeuLeuAlaThrLysLysThrGlyArg                              505560                                                                        LysIleCysLeuAspGlnGlnArgProLeuTyrLysLysIleLeuLys                              65707580                                                                      LysLeuLeuAspGlyAspGluSer                                                      85                                                                            (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     AspValLeuAlaArgValSerAlaGluLeuArgCysGlnCysIleAsn                              151015                                                                        ThrHisSerThrProPheHisProLysPheIleLysGluLeuArgVal                              202530                                                                        IleGluSerGlyProHisCysGluAsnSerGluIleIleValLysLeu                              354045                                                                        ValAsnGlyLysGluValCysLeuAspProLysGluLysTrpValGln                              505560                                                                        LysValValGlnIlePheLeuLysArgThrGluLysGlnGlnGlnGln                              65707580                                                                      Gln                                                                           (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     SerProIleGluAlaAlaGluAlaAlaValValArgGluLeuArgCys                              151015                                                                        MetCysLeuThrThrThrProGlyIleHisProLysMetIleSerAsp                              202530                                                                        LeuGlnValIleProAlaGlyProGlnCysSerLysAlaGluValIle                              354045                                                                        AlaThrLeuLysAsnGlyLysGluValCysLeuAspProLysAlaPro                              505560                                                                        LeuIleLysLysIleValGlnLysMetLeuAspSerGlyLysLysLys                              65707580                                                                      Asn                                                                           (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     AlaValLeuThrArgIleGlyThrGluLeuArgCysGlnCysIleLys                              151015                                                                        ThrHisSerThrProPheHisProLysPheIleLysGluLeuArgVal                              202530                                                                        IleGluSerGlyProHisCysAlaAsnSerGluIleIleValLysLeu                              354045                                                                        ValAspGlyArgGluLeuCysLeuAspProLysGluLysTrpValGln                              505560                                                                        LysValValGlnIlePheLeuLysArgAlaGluGlnGlnGluSer                                 657075                                                                        (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     AlaLeuThrGluLeuArgCysGlnCysLeuGlnThrValGlnGlyIle                              151015                                                                        HisLeuLysSerIleGlnSerLeuLysValLeuSerProGlyProHis                              202530                                                                        CysAlaGlnThr                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     ArgLeuAlaThrGlyAlaProValAlaAsnGluLeuArgCysGlnCys                              151015                                                                        LeuGlnThrHisThrGlyValHisLeuLysAsnIleGluSerLeuLys                              202530                                                                        ValThrProProGlyProHisCysThrGlnThrGluValIleAlaThr                              354045                                                                        LeuLysAsnGlyGlnGluAlaCysLeuAsnProGluAlaProMetVal                              505560                                                                        GlnLysIleValGlnLysMetLeuLysSerGlyIleArgLys                                    657075                                                                        (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     AlaProValAlaAsnGluLeuArgCysGlnCysLeuGlnThrValAla                              151015                                                                        GlyIleHisPheLysAsnIleGlnSerLeuLysValMetProProGly                              202530                                                                        ProHisCysThrGlnThrGluValIleAlaThrLeuLysAsnGlyArg                              354045                                                                        GluAlaCysLeuAspProGluAlaProMetValGlnLysIleValGln                              505560                                                                        LysMetLeuLysGlyValProLys                                                      6570                                                                          (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 76 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     ValThrArgAlaSerProGluGluSerAspGlyAspLeuSerCysVal                              151015                                                                        CysValLysThrSerSerSerArgIleHisLeuLysArgIleThrSer                              202530                                                                        LeuGluValIleLysAlaGlyProHisCysAlaValProGlnLeuIle                              354045                                                                        AlaThrLeuLysAsnGlySerLysIleCysLeuAspArgGlnValPro                              505560                                                                        LeuTyrLysLysIleIleLysLysLeuLeuGluSer                                          657075                                                                        (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     ArgLeuAlaThrGlyAlaProIleAlaAsnGluLeuArgCysGlnCys                              151015                                                                        LeuGlnThrMetAlaGlyIleHisLeuLysAsnIleGlnSerLeuLys                              202530                                                                        ValLeuProSerGlyProHisCysThrGlnThrGluValIleAlaThr                              354045                                                                        LeuLysAsnGlyArgGluAlaCysLeuAspProGluAlaProLeuVal                              505560                                                                        GlnLysIleValGlnLysMetLeuLysGlyValProLys                                       657075                                                                        (2) INFORMATION FOR SEQ ID NO: 42:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     AlaValValAlaSerGluLeuArgCysGlnCysLeuLysThrLeuPro                              151015                                                                        ArgValAspPheLysAsnIleGlnSerLeuSerValThrProProGly                              202530                                                                        ProHisCysAlaGlnThrGluValIleAlaThrLeuLysGlyGlyGln                              354045                                                                        LysValCysLeuAspProGluAlaProLeuValGlnLysIleIleGln                              505560                                                                        LysIleLeuAsnLysGlyLysAlaAsn                                                   6570                                                                          (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     ThrLeuValIleArgAsnAlaArgCysSerCysIleSerThrSerArg                              151015                                                                        GlyThrIleHisTyrLysSerLeuLysAspLeuLysGlnPheAlaPro                              202530                                                                        SerProAsnCysAsnLysThrGluIleIleAlaThrLeuLysAsnGly                              354045                                                                        AspGlnThrCysLeuAspProAspSerAlaAsnValLysLysLeuMet                              505560                                                                        LysGluTrpGluLysLysIleAsnGlnLysLysLysGln                                       657075                                                                        (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     IleProLeuAlaArgThrValArgCysAsnCysIleHisIleAspAsp                              151015                                                                        GlyProValArgMetArgAlaIleGlyLysLeuGluIleIleProAla                              202530                                                                        SerLeuSerCysProArgValGluIleIleAlaThrMetLysLysAsn                              354045                                                                        AspGluGlnArgCysLeuAsnProGluSerLysThrIleLysAsnLeu                              505560                                                                        MetLysAlaPheSerGlnLysArgSerLysArgAlaPro                                       657075                                                                        (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 82 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     ArgThrLeuValLysMetGlyAsnGluLeuArgCysGlnCysIleSer                              151015                                                                        ThrHisSerLysPheIleHisProLysSerIleGlnAspValLysLeu                              202530                                                                        ThrProSerGlyProHisCysLysAsnValGluIleIleAlaThrLeu                              354045                                                                        LysAspGlyArgGluValCysLeuAspProThrAlaProTrpValGln                              505560                                                                        LeuIleValLysAlaLeuMetAlaLysAlaGlnLeuAsnSerAspAla                              65707580                                                                      ProLeu                                                                        __________________________________________________________________________

We claim:
 1. A substantially pure mammalian granulocyte chemotacticprotein-2 (GCP-2).
 2. A GCP-2 protein as defined in claim 1 which ismammalian GCP-2.
 3. A GCP-2 protein as defined in claim 2, having anamino acid sequence of SEQ ID NO:
 14. 4. A CCP-2 protein as defined inclaim 2, having an amino acid sequence of SEQ ID NO:
 15. 5. A GCP-2protein as defined in claim 3 wherein said protein is a variant thereofthat shares no less than about 80% sequence identity of SEQ ID NO:14 andthat retains receptor binding activity.
 6. A GCP-2 protein as defined inclaim 5, which is an amino-terminally truncated variant thereof.
 7. AGCP-2 protein as defined in claim 6, wherein the amino acid sequence ofthe amino-termninus of said variant corresponds to SEQ. ID NO:
 3. 8. AGCP-2 protein as defined in claim 6, wherein the amino acid sequence ofthe amino-terminus of said variant corresponds to SEQ. ID NO:
 4. 9. AGCP-2 protein as defined in claim 6, wherein the amino acid sequence ofamino-terminus of said variant corresponds to SEQ. ID NO:
 5. 10. A GCP-2protein as defined in claim 4 wherein said protein is a variant thereofthat shares no less than about 80% sequence identity of SEQ ID NO:15 andthat retains receptor binding activity.
 11. A GCP-2 protein as definedin claim 10, which is an amino-terminally truncated variant thereof. 12.A GCP-2 protein as defined in claim 11, wherein the amino acid sequenceof the amino-terminus of said variant corresponds to SEQ. ID NO:
 16. 13.A GCP-2 protein as defined in claim 11, wherein the amino acid sequenceof the amino-terminus of said variant corresponds to SEQ. ID NO:
 17. 14.A GCP-2 protein as defined in claim 11, wherein the amino acid sequenceof the amino-terminus of said variant corresponds to SEQ. ID NO:
 18. 15.A GCP-2 protein as defined in claim 1, which is a distint fragment ofGCP-2 that retains receptor binding activity.
 16. A pharmaceuticalcomposition comprising a therapeutically effective amount of a GCP-2protein and a pharmaceutically acceptable carrier.
 17. An isolatedpolynucleotide encoding a GCP-2 protein.
 18. A vector havingincorporated therein a polynucleotide encoding a GCP-2 protein.
 19. Ahost cell that has been engineered genetically to produce a GCP-2protein, the cell having incorporated expressibly therein heterologousDNA encoding said protein.
 20. A method of producing a GCP-2 proteincomprising the step of culturing genetically engineered cells which haveincorporated expressibly therein a heterologous DNA sequence encodingsaid protein.